肾脾阳虚致衰的机制研究
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摘要
目的：本研究立足肾脾相关理论,以肌注氢化可的松和灌胃大黄水煎液复制的肾脾阳虚大鼠模型为平台,观察模型大鼠的衰老变化,以肾脾阳虚对Klotho蛋白表达的影响为切入点,探讨Klotho蛋白及其调控的INS/IGF-1/PI3K/Akt信号转导通路与肾脾阳虚致衰的相关机制,并观察桂附理中丸对肾脾阳虚模型大鼠衰老指标的影响。运用现代研究手段检测其SOD活力、MDA含量,Klotho、Akt、Foxo3a三种基因mRNA及蛋白表达等衰老相关指标,旨在从Klotho蛋白表达调控细胞内的信号转导通路初步阐明肾脾阳虚致衰老的机理。
方法：本课题采用8周龄SD大鼠40只,雄性,随机分为正常对照组、模型组、维生素E组和桂附理中丸组四组。除正常对照组外,其余各组大鼠均采用肌注氢化可的松和灌胃大黄水煎液复制肾脾阳虚大鼠模型。正常对照组大鼠每天上午8：00肌肉注射生理盐水1ml/200g体重,同时灌胃生理盐水2ml/200g体重,下午14：00灌胃生理盐水2ml/200g体重；其余组大鼠上午8：00肌肉注射氢化可的松1ml/200g体重(药物浓度为5mg/ml),同时灌胃大黄水煎液2ml/200g体重(药物浓度为0.4g/ml),下午14：00模型组灌胃生理盐水2ml/200g体重,维生素E组灌胃维生素E混悬液2m1/200g体重(药物浓度为0.0018g/ml),桂附理中丸组灌胃桂附理中丸混悬液2ml/200g体重(药物浓度为0.162g/ml),共30天。从造模结束次日起,所有实验大鼠取24小时尿液,腹主动脉采血后,再剥离大脑海马组织进行相关指标检测：①检测24小时尿17-OH-CS及血清睾酮(T)含量；②检测血清胃泌素、D—木糖含量；③观察海马组织的超微结构改变；④检测血清超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量；⑤取海马CA1区小块组织制备单细胞悬液,流式细胞仪检测大脑海马组织细胞周期,分析各时相区的百分率以明确细胞老化率；⑥实时荧光定量PCR检测信号转导通路(Klotho-INS/IGF-1-PI3K-Akt-FOXO)中Klotho、Akt、Foxo3a三种衰老相关基因的mRNA表达；⑦蛋白免疫印迹及免疫组织化学法染色检测信号转导通路(Klotho-INS/IGF-1-PI3K-Akt-FOXO)中Klotho、Akt、Foxo3a三种基因的蛋白表达。
结果：①24小时尿液、血清指标检测：与正常对照组比较,模型组大鼠尿液17-OH-CS、血清T、胃泌素、D—木糖含量显著降低(P<0.01),SOD活力显著下降(P<0.01),血清MDA含量显著上升(P<0.01)；与模型组比较,维生素E组大鼠尿17-OH-CS、血清T、胃泌素、D—木糖含量及SOD活力均明显升高(P<0.05),血清MDA含量明显下降(P<0.05),桂附理中丸组大鼠血清T、胃泌素、D—木糖含量均显著升高(P<0.01),尿17-OH-CS、血清SOD活力明显升高(P<0.05),血清MDA含量明显下降(P<0.05)；与维生素E组比较,桂附理中丸组大鼠尿17-OH-CS、血清T、胃泌素、D—木糖含量明显升高(P<0.05),血清SOD活力明显升高(P<0.05)。
②大脑海马组织指标检测：与正常对照组比较,模型组大脑海马细胞器减少,大量脂褐素沉积及海马神经元萎缩、细胞突触间隙加宽,细胞老化率明显上升；Klotho调控的细胞信号转导通路(Klotho-INS/IGF-1-PI3K-Akt-FOXO)中KlothomRNA表达水平明显降低(P<0.05),Foxo3amRNA表达水平明显升高(P<0.05),AktmRNA表达水平显著升高(P<0.01),Klotho阳性细胞的平均灰度值显著降低(P<0.01),P-Foxo3a阳性细胞的平均灰度值显著升高(P<0.01),P-Akt阳性细胞的平均灰度值明显升高(P<0.05)；与模型组比较,维生素E组及桂附理中丸组大脑海马细胞器增多,脂褐素沉积减少,较少见到海马神经元萎缩、细胞突触间隙加宽,细胞老化率明显下降,维生素E组大鼠KlothomRNA表达水平、平均灰度值明显升高(P<0.05), Akt、Foxo3amRNA表达水平,P-Akt、P-Foxo3a阳性细胞的平均灰度值均明显降低(P<0.05),桂附理中丸组大鼠KlothomRNA表达水平明显升高(P<0.05),Akt、Foxo3amRNA表达水平,P-Akt阳性细胞的平均灰度值均明显降低(P<0.05),Klotho阳性细胞的平均灰度值显著升高(P<0.01),P-Foxo3a阳性细胞的平均灰度值显著降低(P<0.01)；与维生素E组比较,桂附理中丸组大脑海马细胞器增多,很少见到脂褐素沉积减少,海马神经元萎缩、细胞突触间隙加宽,细胞老化率明显下降,Klotho阳性细胞的平均灰度值明显升高(P<0.05), AktmRNA的表达水平、P-Foxo3a阳性细胞的平均灰度值均明显降低(P<0.05)。
结论：①采用肌注氢化可的松和灌胃大黄水煎液复合方法复制肾脾阳虚大鼠模型,模型大鼠的一般情况符合肾阳虚、脾阳虚证的诊断标准,且模型大鼠24小时尿17-OH-CS、血清T、血清胃泌素、D-木糖含量均显著下降,表明肾脾阳虚大鼠模型复制成功。
②模型组大鼠海马cA1区细胞浆内细胞器较少,有大量脂褐素沉积,神经元细胞突触间隙加宽、神经元萎缩、水样变性等现象；血清SOD活力显著下降、MDA含量明显升高及细胞老化率上升,表明肾脾阳虚模型大鼠存在衰老变化。
③温补肾脾方药—桂附理中丸可以激发机体抗衰老基因Klotho表达,促进FOXO的核迁移,直接调控并且上调SOD2的表达,增强抗氧化酶活力,修复机体受损组织器官,从而延缓肾脾阳虚导致衰老的进程。
④肾脾阳虚模型大脑海马组织Klotho蛋白表达降低,对INS-IGF-1-PI3K-Akt信号级联的抑制减弱,信号转导通路下游Akt、 Foxo3a的磷酸化反应增强,磷酸化Akt、Foxo3a蛋白表达均增多,致使FOXO的核迁移减少,SOD2(MnSOD)的表达下调,超氧化物对机体的损伤增加,从而加速机体衰老。
本研究首次采用肌注氢化可的松和灌胃大黄水煎液复合方法成功复制肾脾阳虚大鼠模型,温补肾脾方药——桂附理中丸可以延缓肾脾阳虚导致衰老的进程。肾脾阳虚模型大鼠由于其海马组织Klotho蛋白表达降低,进而使其下游的INS-IGF-1信号级联抑制减弱,信号转导通路下游Akt、Foxo3a的磷酸化反应增强,磷酸化Akt、Foxo3a蛋白表达增多,FOXO从细胞质移入细胞核减少,SOD2(MnSOD)的表达下调,超氧化物对机体的损害增加,从而加速机体衰老。由此可知,Klotho蛋白低表达是导致肾脾阳虚模型大鼠衰老的可能机制。
Objects:This research based on the related theoretical of kidney and spleen,using the intramuscular injection of hydrocortisone and intrgastric administration of decoction liquid of Da huang established deficiency of kidney and spleen-Yan animal models as a platform, observe changes of model rats aging, take the kidney and spleen-Yang deficiency to the protein expression influence of klotho as the breakthrough point, discuss related mechanism which of the klotho protein and its regulation and control of INS/IGF-1/PI3K/Akt signal transduction pathway and the kidney and spleen-Yang deficiency responsible for aging processes, to observe the effectiveness of the Guifulizhongwan to deficiency of kidney and spleen-Yan animal models on the aspects of aging impact indicators. Application of modern research tools detect its SOD, MDA, klotho, Akt, Foxo3a protein expression and other aging related indexes. In order to preliminary clarify the mechanism of kidney and spleen-Yang deficiency induced aging from the Klotho protein expression regulation of intracellular signal transduction pathways.
Methods:Totally40SD male mice at the age of eight weeks, these mice were randomly divided into normal control group, model group,Vitamin E group, Guifulizhongwan group,ten normal mice in each group, which were separately given medicine. Excluding normal control group, the mice were injected with hydrocortisone and administered with decoction liquid of Da huang for established deficiency of kidney and spleen-Yan disease models. While, normal control group were injected physiologic saline with1ml/200g and administered physiologic saline with2ml/200g at8:00am, it was administered physiologic saline with2ml/200g at2:00pm, the rest of the group were injected hydrocortisone with lml/200g (Drug concentration was5mg/ml) and administered decoction liquid of Da huang with2ml/200g (Drug concentration was0.4g/ml) at8:00am, model group was administered physiologic saline with2ml/200g, Vitamin E group was administered was given liquid mixed suspension of Vitamin E with2ml/200g(Drug concentration was0.0018g/ml) and Guifulizhongw-an group was given liquid mixed suspension of Guifulizhongwan with2ml/200g(Drug concentration was0.162g/ml) at2:00pm, once a day, the procedures lasted for30days.Then, taking24hours of urine, blooding abdominal aortic dissection after stripping the hippocampus of the brain for the index of relevant examination.①Detection of24-hour urinary17-OH-CS and serum testosterone content;②The measurement of serum gastrin and Xylose content;③Observation of ultrastructure of hippocam-pus changes;④Detection of serum superoxide dismutase activity and the malonaldehyde content;⑤Taking the hippocampus CA1area small piece of tissue preparation single-celled levitation liquid, testing the brain hippocampus cells cycles by flow cytometry, analysis of the percentage of each phase in order to clear cell of aging rate;⑥Detection of Klotho, Akt and Foxo3a three age-related genes mRNA expression in the signal transduction pathway (Klotho-INS/IGF-1-PI3K-Akt-FOXO) by real-time fluorescence quantitative polymerase chain reaction (PCR) method;⑦Adopting protein immune imprinting (Western blotting) method and immune histochemistry staining method to detection of klotho, Akt, Foxo3a three kind of gene protein expressions in the signal transduction pathway (Klotho-INS/IGF-1-PI3K-Akt-FOXO).
Results:24hours urine and serum indicators testing:compare with the normal control group, the model group mice whice of urinary17-OH-CS, serum testosterone, serum gastrin, D-Xylose content and serum SOD activity were significant decreased (P<0.01), MDA content was dramatically increased (P<0.01); compare with the model control group, the Vitamin E group mice whice of urinary17-OH-CS, serum testosterone, serum gastrin, D-Xylose content and serum SOD activity were obviously increased(P<0.05), MDA content was obviously decreased(P<0.05), the Guifulizhongwan group mice whice of serum testosterone, serum gastrin and D-Xylose content were dramatically increased (P<0.01), urinary17-OH-CS content and serum SOD activity were obviously increased (P<0.05), MDA content was obviously decreased (P<0.05); compare with the Vitamin E group, the Guifulizhongwan group mice whice of urinary17-OH-CS, serum testosterone, serum gastrin, D-Xylose content and serum SOD activity were obviously increased (P<0.05).
②Detection of brain hippocampus indexes:compare with the normal control group, the model group mice whice of hippocampal organization organelles were reduced, the neuron atrophy, neurons synaptic gap widening, cell cycle had arrested in G1phase, the cell aging rate had obviously rised;KlothomRNA expression had obviously decreased (P<0.05), Foxo3amRNA expression had obviously increased(P<0.05), AktmRNA expression had significant increased (P<0.05) in signal transduction pathway (Klotho-INS/IGF-1-PI3K-Akt-FOXO), Klotho-positive cells significantly reduced the average gray level value, P-Foxo3a positive cells significantly increased, the average gray level value(P<0.01),P-Akt positive cells obviously increased, the average gray level value (P<0.05); compare with the model control group, the Vitamin E and Guifulizhongwan group group mice whice of hippocampal organization organelles were increased, withers as rarely as the neuron atrophy, neurons synaptic gap widening, the cell aging rate had obviously droped; the Vitamin E group mice whice of KlothomRNA expression and the average gray level value of Klotho-positive cells had obviously increased (P<0.05), the level of expression of Akt and Foxo3amRNA, P-Akt, P-Foxo3a-positive cells mean gray value had obviously reduced (P<0.05), the Guifulizhongwan group mice whice of KlothomRNA expression levels had obviously increased (P<0.05), levels of expression of Akt and Foxo3amRNA and mean gray value of P-Akt-positive cells had obviously reduced(P<0.05), Klotho-positive cells mean gray value had significantly rised(P<0.01), P-Foxo3a-positive cells mean gray value had significantly reduced (P<0.01); compare with the Vitamin E group, the Guifulizhongwan group mice whice of hippocampal organization organelles were increased, it hardly to see withers as rarely as the neuron atrophy, neurons synaptic gap widening, the cell aging rate had obviously droped, Klotho-positive cells mean gray value had obviously increased (P<0.05), the levels of expression of AktmRNA and P-Foxo3a-positive cells mean gray value had obviously reduced (P<0.05).
Conclusion:①The model group of mouse's ordinary circumstances, according with kidney-Yang deficiency, spleen-Yang deficiency syndrome diagnostic criteria; it were significantly lower decreased24-hour urinary17-OH-CS, serum testosterone content、gastrin and Xylose content in model group, using injected with hydrocortisone and administered with decoction liquid of Da huang had established deficiency of kidney and spleen-Yan disease models successfully.
②The model group of mouse's hippocampal organization organelles were reduced,neurons synaptic gap widening, the neuron atrophy, hydropic degeneration; serum SOD activity was decreased, MDA content was increased; Hippocampus of the brain organization cell cycle had arrested in the highest proportion of G1phase, the cell aging rate had rised, the deficiency of kidney and spleen-Yan disease models mice has appeared aging.
￤arming and recuperating kidney-Guifulizhongwan can increased the body's anti-aging gene klotho expression, promoed FOXO nuclear migration, increased the expression SOD2, prompted an increase in antioxidant enzyme synthesis, repaired organism damage organization organ, thus the postponement deficiency of kidney and spleen-Yan causes the aging advancement.
④The deficiency of kidney and spleen-Yan disease models mice of the brain hippocampus of klotho protein expression was reduced, inhibition of INS/IGF-1signal cascade was weaken, thereby enhanced the phosphorylation of Akt and Foxo3a in downstream of the signal transduction pathway expression of phosphorylated Akt and foxo3a protein were increased, reduced FOXO nuclear transfer, declined the expression SOD2, resulting in ability to clear the oxygen free radical was reduced, increasing in superoxide damage to the body, to speed up the body's aging.
This study was the first to using injected with hydrocortisone and administered with decoction liquid of Da huang had established deficiency of kidney and spleen-Yan disease models successfully, Guifulizhongwan can postpone deficiency of kidney and spleen-Yan causes the aging advancement, because the deficiency of kidney and spleen-Yan disease models mice of the brain hippocampus of klotho protein expression was reduced, inhibition of INS/IGF-1signal cascade was weaken, reduced FOXO nuclear transfer, declined the expression SOD2, to speed up the body's aging. It can be seen, the mechanism which of kidney and spleen-Yan deficiency responsible for aging may be because the brain hippocampus of klotho protein expression was reduced.
方法：本课题采用8周龄SD大鼠40只,雄性,随机分为正常对照组、模型组、维生素E组和桂附理中丸组四组。除正常对照组外,其余各组大鼠均采用肌注氢化可的松和灌胃大黄水煎液复制肾脾阳虚大鼠模型。正常对照组大鼠每天上午8：00肌肉注射生理盐水1ml/200g体重,同时灌胃生理盐水2ml/200g体重,下午14：00灌胃生理盐水2ml/200g体重；其余组大鼠上午8：00肌肉注射氢化可的松1ml/200g体重(药物浓度为5mg/ml),同时灌胃大黄水煎液2ml/200g体重(药物浓度为0.4g/ml),下午14：00模型组灌胃生理盐水2ml/200g体重,维生素E组灌胃维生素E混悬液2m1/200g体重(药物浓度为0.0018g/ml),桂附理中丸组灌胃桂附理中丸混悬液2ml/200g体重(药物浓度为0.162g/ml),共30天。从造模结束次日起,所有实验大鼠取24小时尿液,腹主动脉采血后,再剥离大脑海马组织进行相关指标检测：①检测24小时尿17-OH-CS及血清睾酮(T)含量；②检测血清胃泌素、D—木糖含量；③观察海马组织的超微结构改变；④检测血清超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量；⑤取海马CA1区小块组织制备单细胞悬液,流式细胞仪检测大脑海马组织细胞周期,分析各时相区的百分率以明确细胞老化率；⑥实时荧光定量PCR检测信号转导通路(Klotho-INS/IGF-1-PI3K-Akt-FOXO)中Klotho、Akt、Foxo3a三种衰老相关基因的mRNA表达；⑦蛋白免疫印迹及免疫组织化学法染色检测信号转导通路(Klotho-INS/IGF-1-PI3K-Akt-FOXO)中Klotho、Akt、Foxo3a三种基因的蛋白表达。
结果：①24小时尿液、血清指标检测：与正常对照组比较,模型组大鼠尿液17-OH-CS、血清T、胃泌素、D—木糖含量显著降低(P<0.01),SOD活力显著下降(P<0.01),血清MDA含量显著上升(P<0.01)；与模型组比较,维生素E组大鼠尿17-OH-CS、血清T、胃泌素、D—木糖含量及SOD活力均明显升高(P<0.05),血清MDA含量明显下降(P<0.05),桂附理中丸组大鼠血清T、胃泌素、D—木糖含量均显著升高(P<0.01),尿17-OH-CS、血清SOD活力明显升高(P<0.05),血清MDA含量明显下降(P<0.05)；与维生素E组比较,桂附理中丸组大鼠尿17-OH-CS、血清T、胃泌素、D—木糖含量明显升高(P<0.05),血清SOD活力明显升高(P<0.05)。
②大脑海马组织指标检测：与正常对照组比较,模型组大脑海马细胞器减少,大量脂褐素沉积及海马神经元萎缩、细胞突触间隙加宽,细胞老化率明显上升；Klotho调控的细胞信号转导通路(Klotho-INS/IGF-1-PI3K-Akt-FOXO)中KlothomRNA表达水平明显降低(P<0.05),Foxo3amRNA表达水平明显升高(P<0.05),AktmRNA表达水平显著升高(P<0.01),Klotho阳性细胞的平均灰度值显著降低(P<0.01),P-Foxo3a阳性细胞的平均灰度值显著升高(P<0.01),P-Akt阳性细胞的平均灰度值明显升高(P<0.05)；与模型组比较,维生素E组及桂附理中丸组大脑海马细胞器增多,脂褐素沉积减少,较少见到海马神经元萎缩、细胞突触间隙加宽,细胞老化率明显下降,维生素E组大鼠KlothomRNA表达水平、平均灰度值明显升高(P<0.05), Akt、Foxo3amRNA表达水平,P-Akt、P-Foxo3a阳性细胞的平均灰度值均明显降低(P<0.05),桂附理中丸组大鼠KlothomRNA表达水平明显升高(P<0.05),Akt、Foxo3amRNA表达水平,P-Akt阳性细胞的平均灰度值均明显降低(P<0.05),Klotho阳性细胞的平均灰度值显著升高(P<0.01),P-Foxo3a阳性细胞的平均灰度值显著降低(P<0.01)；与维生素E组比较,桂附理中丸组大脑海马细胞器增多,很少见到脂褐素沉积减少,海马神经元萎缩、细胞突触间隙加宽,细胞老化率明显下降,Klotho阳性细胞的平均灰度值明显升高(P<0.05), AktmRNA的表达水平、P-Foxo3a阳性细胞的平均灰度值均明显降低(P<0.05)。
结论：①采用肌注氢化可的松和灌胃大黄水煎液复合方法复制肾脾阳虚大鼠模型,模型大鼠的一般情况符合肾阳虚、脾阳虚证的诊断标准,且模型大鼠24小时尿17-OH-CS、血清T、血清胃泌素、D-木糖含量均显著下降,表明肾脾阳虚大鼠模型复制成功。
②模型组大鼠海马cA1区细胞浆内细胞器较少,有大量脂褐素沉积,神经元细胞突触间隙加宽、神经元萎缩、水样变性等现象；血清SOD活力显著下降、MDA含量明显升高及细胞老化率上升,表明肾脾阳虚模型大鼠存在衰老变化。
③温补肾脾方药—桂附理中丸可以激发机体抗衰老基因Klotho表达,促进FOXO的核迁移,直接调控并且上调SOD2的表达,增强抗氧化酶活力,修复机体受损组织器官,从而延缓肾脾阳虚导致衰老的进程。
④肾脾阳虚模型大脑海马组织Klotho蛋白表达降低,对INS-IGF-1-PI3K-Akt信号级联的抑制减弱,信号转导通路下游Akt、 Foxo3a的磷酸化反应增强,磷酸化Akt、Foxo3a蛋白表达均增多,致使FOXO的核迁移减少,SOD2(MnSOD)的表达下调,超氧化物对机体的损伤增加,从而加速机体衰老。
本研究首次采用肌注氢化可的松和灌胃大黄水煎液复合方法成功复制肾脾阳虚大鼠模型,温补肾脾方药——桂附理中丸可以延缓肾脾阳虚导致衰老的进程。肾脾阳虚模型大鼠由于其海马组织Klotho蛋白表达降低,进而使其下游的INS-IGF-1信号级联抑制减弱,信号转导通路下游Akt、Foxo3a的磷酸化反应增强,磷酸化Akt、Foxo3a蛋白表达增多,FOXO从细胞质移入细胞核减少,SOD2(MnSOD)的表达下调,超氧化物对机体的损害增加,从而加速机体衰老。由此可知,Klotho蛋白低表达是导致肾脾阳虚模型大鼠衰老的可能机制。
Objects:This research based on the related theoretical of kidney and spleen,using the intramuscular injection of hydrocortisone and intrgastric administration of decoction liquid of Da huang established deficiency of kidney and spleen-Yan animal models as a platform, observe changes of model rats aging, take the kidney and spleen-Yang deficiency to the protein expression influence of klotho as the breakthrough point, discuss related mechanism which of the klotho protein and its regulation and control of INS/IGF-1/PI3K/Akt signal transduction pathway and the kidney and spleen-Yang deficiency responsible for aging processes, to observe the effectiveness of the Guifulizhongwan to deficiency of kidney and spleen-Yan animal models on the aspects of aging impact indicators. Application of modern research tools detect its SOD, MDA, klotho, Akt, Foxo3a protein expression and other aging related indexes. In order to preliminary clarify the mechanism of kidney and spleen-Yang deficiency induced aging from the Klotho protein expression regulation of intracellular signal transduction pathways.
Methods:Totally40SD male mice at the age of eight weeks, these mice were randomly divided into normal control group, model group,Vitamin E group, Guifulizhongwan group,ten normal mice in each group, which were separately given medicine. Excluding normal control group, the mice were injected with hydrocortisone and administered with decoction liquid of Da huang for established deficiency of kidney and spleen-Yan disease models. While, normal control group were injected physiologic saline with1ml/200g and administered physiologic saline with2ml/200g at8:00am, it was administered physiologic saline with2ml/200g at2:00pm, the rest of the group were injected hydrocortisone with lml/200g (Drug concentration was5mg/ml) and administered decoction liquid of Da huang with2ml/200g (Drug concentration was0.4g/ml) at8:00am, model group was administered physiologic saline with2ml/200g, Vitamin E group was administered was given liquid mixed suspension of Vitamin E with2ml/200g(Drug concentration was0.0018g/ml) and Guifulizhongw-an group was given liquid mixed suspension of Guifulizhongwan with2ml/200g(Drug concentration was0.162g/ml) at2:00pm, once a day, the procedures lasted for30days.Then, taking24hours of urine, blooding abdominal aortic dissection after stripping the hippocampus of the brain for the index of relevant examination.①Detection of24-hour urinary17-OH-CS and serum testosterone content;②The measurement of serum gastrin and Xylose content;③Observation of ultrastructure of hippocam-pus changes;④Detection of serum superoxide dismutase activity and the malonaldehyde content;⑤Taking the hippocampus CA1area small piece of tissue preparation single-celled levitation liquid, testing the brain hippocampus cells cycles by flow cytometry, analysis of the percentage of each phase in order to clear cell of aging rate;⑥Detection of Klotho, Akt and Foxo3a three age-related genes mRNA expression in the signal transduction pathway (Klotho-INS/IGF-1-PI3K-Akt-FOXO) by real-time fluorescence quantitative polymerase chain reaction (PCR) method;⑦Adopting protein immune imprinting (Western blotting) method and immune histochemistry staining method to detection of klotho, Akt, Foxo3a three kind of gene protein expressions in the signal transduction pathway (Klotho-INS/IGF-1-PI3K-Akt-FOXO).
Results:24hours urine and serum indicators testing:compare with the normal control group, the model group mice whice of urinary17-OH-CS, serum testosterone, serum gastrin, D-Xylose content and serum SOD activity were significant decreased (P<0.01), MDA content was dramatically increased (P<0.01); compare with the model control group, the Vitamin E group mice whice of urinary17-OH-CS, serum testosterone, serum gastrin, D-Xylose content and serum SOD activity were obviously increased(P<0.05), MDA content was obviously decreased(P<0.05), the Guifulizhongwan group mice whice of serum testosterone, serum gastrin and D-Xylose content were dramatically increased (P<0.01), urinary17-OH-CS content and serum SOD activity were obviously increased (P<0.05), MDA content was obviously decreased (P<0.05); compare with the Vitamin E group, the Guifulizhongwan group mice whice of urinary17-OH-CS, serum testosterone, serum gastrin, D-Xylose content and serum SOD activity were obviously increased (P<0.05).
②Detection of brain hippocampus indexes:compare with the normal control group, the model group mice whice of hippocampal organization organelles were reduced, the neuron atrophy, neurons synaptic gap widening, cell cycle had arrested in G1phase, the cell aging rate had obviously rised;KlothomRNA expression had obviously decreased (P<0.05), Foxo3amRNA expression had obviously increased(P<0.05), AktmRNA expression had significant increased (P<0.05) in signal transduction pathway (Klotho-INS/IGF-1-PI3K-Akt-FOXO), Klotho-positive cells significantly reduced the average gray level value, P-Foxo3a positive cells significantly increased, the average gray level value(P<0.01),P-Akt positive cells obviously increased, the average gray level value (P<0.05); compare with the model control group, the Vitamin E and Guifulizhongwan group group mice whice of hippocampal organization organelles were increased, withers as rarely as the neuron atrophy, neurons synaptic gap widening, the cell aging rate had obviously droped; the Vitamin E group mice whice of KlothomRNA expression and the average gray level value of Klotho-positive cells had obviously increased (P<0.05), the level of expression of Akt and Foxo3amRNA, P-Akt, P-Foxo3a-positive cells mean gray value had obviously reduced (P<0.05), the Guifulizhongwan group mice whice of KlothomRNA expression levels had obviously increased (P<0.05), levels of expression of Akt and Foxo3amRNA and mean gray value of P-Akt-positive cells had obviously reduced(P<0.05), Klotho-positive cells mean gray value had significantly rised(P<0.01), P-Foxo3a-positive cells mean gray value had significantly reduced (P<0.01); compare with the Vitamin E group, the Guifulizhongwan group mice whice of hippocampal organization organelles were increased, it hardly to see withers as rarely as the neuron atrophy, neurons synaptic gap widening, the cell aging rate had obviously droped, Klotho-positive cells mean gray value had obviously increased (P<0.05), the levels of expression of AktmRNA and P-Foxo3a-positive cells mean gray value had obviously reduced (P<0.05).
Conclusion:①The model group of mouse's ordinary circumstances, according with kidney-Yang deficiency, spleen-Yang deficiency syndrome diagnostic criteria; it were significantly lower decreased24-hour urinary17-OH-CS, serum testosterone content、gastrin and Xylose content in model group, using injected with hydrocortisone and administered with decoction liquid of Da huang had established deficiency of kidney and spleen-Yan disease models successfully.
②The model group of mouse's hippocampal organization organelles were reduced,neurons synaptic gap widening, the neuron atrophy, hydropic degeneration; serum SOD activity was decreased, MDA content was increased; Hippocampus of the brain organization cell cycle had arrested in the highest proportion of G1phase, the cell aging rate had rised, the deficiency of kidney and spleen-Yan disease models mice has appeared aging.
￤arming and recuperating kidney-Guifulizhongwan can increased the body's anti-aging gene klotho expression, promoed FOXO nuclear migration, increased the expression SOD2, prompted an increase in antioxidant enzyme synthesis, repaired organism damage organization organ, thus the postponement deficiency of kidney and spleen-Yan causes the aging advancement.
④The deficiency of kidney and spleen-Yan disease models mice of the brain hippocampus of klotho protein expression was reduced, inhibition of INS/IGF-1signal cascade was weaken, thereby enhanced the phosphorylation of Akt and Foxo3a in downstream of the signal transduction pathway expression of phosphorylated Akt and foxo3a protein were increased, reduced FOXO nuclear transfer, declined the expression SOD2, resulting in ability to clear the oxygen free radical was reduced, increasing in superoxide damage to the body, to speed up the body's aging.
This study was the first to using injected with hydrocortisone and administered with decoction liquid of Da huang had established deficiency of kidney and spleen-Yan disease models successfully, Guifulizhongwan can postpone deficiency of kidney and spleen-Yan causes the aging advancement, because the deficiency of kidney and spleen-Yan disease models mice of the brain hippocampus of klotho protein expression was reduced, inhibition of INS/IGF-1signal cascade was weaken, reduced FOXO nuclear transfer, declined the expression SOD2, to speed up the body's aging. It can be seen, the mechanism which of kidney and spleen-Yan deficiency responsible for aging may be because the brain hippocampus of klotho protein expression was reduced.
引文
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